Dynamic headspace (DHS) technique: set-up and parameter ... · I Acknowledgements I am very grateful for the opportunity that I have received to accomplish this task in Scentarom.

Dynamic headspace (DHS) technique: set-up and

parameter control for GC/MS analysis of odorant

formulations

Ellen Vercruyssen

Supervisors: Prof. dr. Jo Schaubroeck, dr. Jan Van Biesen

Master's dissertation submitted in order to obtain the academic degree of

Master of Science in de industriële wetenschappen: chemie

Department of Industrial Technology and Construction

Chairman: Prof. dr. Marc Vanhaelst

Faculty of Engineering and Architecture

Academic year 2013-2014

I

Acknowledgements

I am very grateful for the opportunity that I have received to accomplish this task in Scentarom. The

combination of the subject and the entourage at Scentarom resulted in the feeling that this was the

best internship I could ever imagine.

I would like to express my appreciation for Dr. J. Van Biesen because he was always available when I

wanted to discuss the content of my thesis. He was full of involvement, patience and enthusiasm

during my process.

Also my special thanks to Dr. J. Schaubroeck and Dr. G. Diricks for the time they put into reading my

thesis and for the adaptations they’ve suggested.

Last but not least I would like to thank my colleague A. Vermoesen for the support during my

internship and the adjustments she made to my thesis.

II

Abstract

The DHS-technique has been explored as possible tool to obtain additional analytical information on

fragrances and flavours present in a complex matrix. The focus of present work is on mastering the

governing (odour) contamination present as ambient background in a fragrance company, while

performing an in-depth analysis.

Furthermore all experimental parameters, important for the DHS technique itself, have studied and

evaluated in the context of specific problems encountered in the loaded environment of a fragrance

production house.

III

Table of Contents

Acknowledgements .................................................................................................................................. I

Abstract ................................................................................................................................................... II

Table of Contents ................................................................................................................................... III

List of Figures ........................................................................................................................................... V

List of Tables ........................................................................................................................................... VI

List of abbreviations .............................................................................................................................. VII

Introduction ............................................................................................................................................. 1

I Literature/method description ....................................................................................................... 2

I.1 GC/MS ......................................................................................................................................... 2

I.1.1 Gas chromatograph ................................................................................................................. 2

I.1.1.1 Carrier gas ........................................................................................................................ 2

I.1.1.2 Sample introduction ........................................................................................................ 3

I.1.1.2.1 Types of sample introduction in GC ........................................................................ 3

I.1.1.2.2 PTV combined with TDU .......................................................................................... 3

I.1.1.3 Column ............................................................................................................................ 7

I.1.1.4 Detector ........................................................................................................................... 7

I.1.2 The GC/MS Interface ............................................................................................................... 7

I.1.3 MS ............................................................................................................................................ 7

I.1.3.1 Sample inlet ..................................................................................................................... 7

I.1.3.2 Ionization system ............................................................................................................. 8

I.1.3.3 Mass analyzer .................................................................................................................. 8

I.1.3.4 Ion detector ..................................................................................................................... 9

I.1.4 Computer ................................................................................................................................. 9

I.2 Sample preparation ................................................................................................................... 10

I.2.1 Static headspace .................................................................................................................... 10

I.2.2 Dynamic headspace ............................................................................................................... 10

I.2.2.1 Description .................................................................................................................... 10

I.2.2.2 Trapping of components ............................................................................................... 11

I.3 Flow charts ................................................................................................................................ 14

II Experimental part .......................................................................................................................... 16

IV

II.1 Short experimental method descriptions ................................................................................. 16

II.1.1 Blank TDU .......................................................................................................................... 17

II.1.2 Blank DHS .......................................................................................................................... 17

II.1.3 DHS analysis of sample ...................................................................................................... 18

II.2 Contamination ........................................................................................................................... 21

II.2.1 Introduction ....................................................................................................................... 21

II.2.2 Sources where contamination can occur .......................................................................... 21

II.2.3 Measurement of contamination ....................................................................................... 22

II.2.3.1 Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax

liner) ....................................................................................................................................... 22

II.2.3.2 TDU-tube in TDU-tray .................................................................................................... 22

II.2.3.3 PTV-liner in PTV ............................................................................................................. 22

II.2.3.4 TDU-tube in TDU............................................................................................................ 23

II.2.3.5 Unlocking of TDU ........................................................................................................... 24

II.2.3.6 Transporting TDU-tube to DHS carriage ........................................................................ 25

II.2.3.7 Septum of the sample vial ............................................................................................. 26

II.2.3.8 DHS station and DHS carriage ....................................................................................... 27

II.2.3.9 O-rings on TDU-head ..................................................................................................... 27

II.2.3.10 Carrier ........................................................................................................................ 28

II.2.4 Conclusion ......................................................................................................................... 30

II.3 Parameters ................................................................................................................................ 32

II.3.1 Introduction ....................................................................................................................... 32

II.3.2 Test sample ........................................................................................................................ 33

II.3.3 Split ratio R in PTV ............................................................................................................. 33

II.3.4 Sample temperature (Tsample i and Tsample tr) during incubation step and trapping step ..... 35

II.3.5 Total incubation time Δti ................................................................................................... 37

II.3.6 Trapping gas flow F (V=constant) ...................................................................................... 39

II.3.7 Trapping gas volume V (F=constant) ................................................................................. 41

II.3.8 Reproducibility .................................................................................................................. 43

II.4 Comparing DHS analysis and SHS analysis ............................................................................... 45

II.5 Shampoo .................................................................................................................................... 47

Conclusion ............................................................................................................................................. 50

Bibliography ........................................................................................................................................... 52

Attachments .......................................................................................................................................... 53

V

List of Figures

Figure 1-Schematic of GC/MS ................................................................................................................. 2

Figure 2-Sample introduction for DHS: thermal desorption (TDU-PTV-Column) .................................... 4

Figure 3-Structure Tenax TA .................................................................................................................... 4

Figure 4-Pneumatic diagram of the flow of the GC, TDU and PTV during desorption step and injection

step .......................................................................................................................................................... 6

Figure 5-Schematic figuration of electron ionization .............................................................................. 8

Figure 6-Schematic figuration of a quadrupole analyzer ........................................................................ 9

Figure 7-Scheme of an electron multiplier .............................................................................................. 9

Figure 8-DHS device............................................................................................................................... 11

Figure 9-Schematic overview when trapping all components .............................................................. 11

Figure 10-GC/MS with configuration for DHS analysis .......................................................................... 13

Figure 11-Schematic overview of GC/MS and sample preparation ...................................................... 14

Figure 12-Schematic overview of trapping and releasing ..................................................................... 15

Figure 13-Schematic overview of parameters of DHS and GC/MS ....................................................... 20

Figure 14-Figuration of PTV-liner, TDU-tube and sample vial ............................................................... 21

Figure 15-Chromatogram of contamination of TDU-tube (containing tenax) in tray ........................... 22

Figure 16-Results of PTV-liner in PTV .................................................................................................... 23

Figure 17-Results of contamination of TDU-tube in TDU ...................................................................... 24

Figure 18-Chromatogram of contamination when the TDU is open for 45 seconds ............................ 25

Figure 19-Chromatogram of test of transporting TDU-tube to DHS carriage ....................................... 25

Figure 20-Figuration of septum of sample vial ...................................................................................... 26

Figure 21-Result for contamination of septum of the sample vial ....................................................... 26

Figure 22-Results for contamination of blank DHS ............................................................................... 27

Figure 23-Contamination of O-rings on TDU head ................................................................................ 28

Figure 24-Results of contamination of carbohydrate and silica............................................................ 29

Figure 25-Chromatograms of blank TDU ............................................................................................... 30

Figure 26-Total area of test sample and total area of contamination .................................................. 34

Figure 27-Chromatogram of blank analysis of split ratio 1/10, 1/25, 1/50 and 1/100 ......................... 35

Figure 28-Influence of incubation temperature on the relative area and the total area of the

components trapped ............................................................................................................................. 36

Figure 29-Influence of incubation time on the relative area and the total area of the components

trapped .................................................................................................................................................. 38

Figure 30-Influence of trapping gas flow on the relative area and the total area of the components

trapped .................................................................................................................................................. 40

Figure 31-Influence of trapping gas volume on the relative area and the total area of the components

trapped .................................................................................................................................................. 42

Figure 32-Reproducibility of DHS on the absolute area of each component and the total area of all

components trapped ............................................................................................................................. 44

Figure 33-Comparing DHS with SHS on the absolute, relative and total area of the components

trapped .................................................................................................................................................. 46

Figure 34-Chromatograms of shampoo with concentration of 100 % shampoo, 50 % shampoo 50 %

H2O, 50 % shampoo 50 % silica and 25 % shampoo 25 % H2O 50 % silica ........................................... 49

VI

List of Tables

Table 1-Overview sample introduction when using TDU/PTV combination ........................................... 5

Table 2-Overview of the split ratio and the start split ratio .................................................................. 32

Table 3-Overview of the parameters of steps of the DHS and the start parameters ........................... 32

Table 4-Overview of components in test sample .................................................................................. 33

Table 5-Overview of the different split ratios and corresponding concentrations that have been

tested ..................................................................................................................................................... 33

Table 6-Overview of the different incubation temperatures that have been tested ........................... 35

Table 7-Overview of the different incubation times that have been tested ........................................ 37

Table 8-Overview of the different trapping gas flows that have been tested ...................................... 39

Table 9-Overview of the different trapping gas volumes that have been tested ................................. 41

Table 10-Total area of the individual analyses ...................................................................................... 45

VII

List of abbreviations

GC/MS: Gas Chromatography/Mass Spectrometry

GC: Gas Chromatograph

MS: Mass Spectrometer

COC: Cold On Column

PTV: Programmed Temperature Vaporizer

TDU: Thermal Desorption Unit

PDMS: polydimethylsiloxane

FID: Flame Ionisation Detector

TCD: Thermal Conductivity detector

m/z: mass-to-charge ratio

EI: Electron Ionisation

SHS: Static Headspace

DHS: Dynamic Headspace

DC: direct current

RF: radio frequency

VOC: volatile organic component

1

Introduction

Scentarom is a company which produces flavours and fragrances. These flavours and fragrances are

mixtures of different components and these components have a typical odour. The amount of

different components is dependent on the nature of the flavour or fragrance.

Because of the complex nature of the matrix to which these flavours and fragrances are added, there

are no straightforward methods for the analysis of the different components in it. GC/MS is an

analytical instrument with a high sensitivity, but fragrances and flavours cannot be injected directly

in the GC/MS (because of the matrix) and therefore a sample preparation is necessary.

In the open environment within Scentarom there is a strong background odour present and this

odour can cause contamination when using adsorption material during measurements. This

contamination will be examined in this work.

Present work is preceded by Nele Piens’ thesis “Static headspace characterisation of odorants in

complex matrices by hyphenated GC-MS techniques”. The SHS-type performed by Nele Pien is a SHS

with the use of trapping material. The trapping material is placed in a glass jar containing the sample

and then sealed with aluminium foil for a certain period of time at a certain temperature. During this

period of time the volatile components of the sample will be adsorped on the trapping material.

After this period of time the trapping material is analysed through GC/MS.

In this work attention is focussed on dynamic headspace (DHS), as more sensitivity could be

expected. DHS is a dynamic technique in which more parameters have to be explored when

compared to the SHS-type analysis described in the previous paragraph.

2

I Literature/method description

I.1 GC/MS Gas chromatography/mass spectrometry (GC/MS) (figure 1) is a synergetic combination of two

powerful analytical techniques. There is a GC which separates components of a mixture in function of

time, depending on the boiling point and the polarity of these components. After separation, the MS

provides information that helps to identify (structural identification) the components in the mixture.

[1]

Figure 1-Schematic of GC/MS

[2]

I.1.1 Gas chromatograph Gas chromatograph is a separation technique used to analyse volatile substances in the gas phase.

Components are dissolved in a solvent and vaporized when heated in the inlet port (injector), so the

components are in the gas phase. The gas chromatograph uses a carrier gas to sweep the

components to the column, that contains a coating of a stationary phase. Separation of components

is determined by the distribution of each component between the carrier gas and the stationary

phase. A component that has a low interaction with the stationary phase, will elute quickly and

reaches the detector first. A polar component interacts with a polar stationary phase. A non polar

component will interact with a non polar stationary phase. Also the boiling point has an influence on

the elution time of the component. Only components that can be vaporized without decomposition

will be suitable for GC analysis. [1, 2]

I.1.1.1 Carrier gas

Helium is the most commonly used carrier gas. Also nitrogen, argon and hydrogen can be used as

carrier gas. The choice of carrier gas depends upon the desired performance and the detector being

used. The carrier gas must be inert, so it won’t react with the components in the sample. It also may

not be retarded by the stationary phase of the column, so the carrier gas will reach the detector first.

The linear velocity of the carrier gas is an important parameter. It can be determined by injecting the

3

carrier gas and measuring the time from injection to detection (=retention time). The linear velocity

is the retention time divided by the column length. [1, 2]

I.1.1.2 Sample introduction

I.1.1.2.1 Types of sample introduction in GC

There are several types of sample introduction. The most common and oldest technique is the

split/splitless injection. Here the liquid sample is introduced into a heated room by a syringe. The

temperature of this room is normally minimum 50°C higher than the temperature of the column. The

sample will evaporate immediately and will be swept to the column by the carrier gas. The injection

of the sample needs to be done quickly to avoid peak broading. When using a splitless injection every

component of the sample will reach the column. To avoid overloading and contamination the

injection can be done by a split injection. Only a small part of the components will reach the column

when using a split injection.

Another type of sample introduction is the Cold On Column (COC) injection. Here the liquid sample is

introduced into a precolumn by a syringe at a low temperature. The precolumn is an uncoated

column with a wider diameter than the analytical column. This is used to avoid contamination of the

column and to refocus the liquid sample at the entrance of the analytical column. The temperature

of the COC is kept at a constant, near ambient temperature during the injection. After the injection

the GC will heat up. Because the sample is introduced directly into the column there is no

discrimination of volatile components.

The most advanced injection technique of sample introduction is a PTV-injector. PTV stands for

Programmed Temperature Vaporizor injection. The different between a PTV and a COC injection is

that the PTV contains a temperature program that can be activated in the software. PTV can heat up

very quickly. It can also be used as a COC injection. PTV can be used in split or splitless mode. [3]

I.1.1.2.2 PTV combined with TDU

Use of a PTV can be combined with a thermal desorption unit (TDU). TDU is a highly efficient thermal

desorption system. It allows the controlled transfer of thermal desorbed material to an injection

system (like PTV).

When TDU is combined with PTV sample injection (figure 2), the sample introduction device contains

2 units, a PTV and a TDU. Both PTV and TDU have a liner which can contain an adsorption or an

absorption material.

There are several kinds of liners, for example: empty glass liners, empty glass mulitbaffled liners,

liners packed with glass beads or Tenax TA or carbotrap or PDMS (polydimetylsiloxane) foam. The

liner in TDU can also contain a part of the sample itself when the sample is a solid.

In this work tenax TA is used in TDU and PTV. Tenax TA is a porous material with a specific surface

area of 35m2/g. It has a low affinity for water and methanol and adsorbs components in the C5-C20

range. Tenax TA is based on 2,6-diphenylphenylene oxide polymer (figure 3). It is used for it stability

when heated (upper limit of 350°C). The tube of PTV contains less tenax than the tube of TDU. The

tube of PTV is also smaller than the tube of TDU. [3, 4]

4

TDU

PTV

Column

Figure 2-Sample introduction for DHS: thermal desorption (TDU-PTV-Column)

[5]

Figure 3-Structure Tenax TA

[6]

There are 2 steps in the sample introduction when using the PTV in combination with the TDU:

Step 1. Thermal desorption (TDU)

The thermal desorption unit contains a liner that can contain an adsorption material used in the

sample preparation (see chapter I.2 Sample preparation, page 10). During the desorption step, TDU is

heated. While heating, the components will be desorbed from the adsorption material in TDU,

carried by helium gas and trapped on the adsorption material in the cold PTV.

Step 2. Sample injection (PTV)

PTV will be heated. While heating, the trapped components will be desorbed from the tenax in PTV,

carried by helium gas to the column. [3]

TDU and/or PTV can be used in split or splitless mode. There are different possible modes of

operation. The use of which methods depends on the type and concentration of the components

present in the sample. The 2 most important ones are mentioned in table 1. [3]

5

Table 1-Overview sample introduction when using TDU/PTV combination

Mode A B

Step 1 Desorption Splitless Splitless

Step 2 Injection Splitless Split

Software TDU Splitless Splitless

PTV Solvent Venting Solvent Venting

Mode A (splitless desorption, splitless injection):

Mode A is used for a quantitative transfer from TDU to the column. This is used when there has to be

a maximum sensitivity.

Step1: Nearly every component, desorbed from the tenax, will be carried by the helium gas and

trapped on the tenax in the cold PTV.

Step 2: Every component, trapped on the tenax of PTV of Step 1, goes quantitatively to the column.

[3]

For an efficient thermal desorption the flow in the TDU needs to be at least 20 ml/min. This flow is

too high for the column (should be 1 ml/min). The TDU and/or PTV therefore need to be in split

during the desorption step. This is done by choosing “solvent venting” in the software program,

allowing the PTV to be used in split or splitless mode. In solvent venting the valve, regulating the

split, closes during a small defined period of time. [3]

In figure 4 a pneumatic diagram of the GC, TDU and PTV during desorption step and injection step is

shown.

Pneumatic diagram during desorption step

PTV

23 ml/min

3 ml/min

16 ml/min

20 ml/min

1 ml/min

6

Pneumatic diagram during injection step

PV 1: Proportional valve

PV 2: Proportional valve

FS: Flow sensor

PS: Pressure sensor

SPR: Purge flow regulator for the septum purge

TPR: Purge flow regulator for the TDU

SV 3: Valve split/splitless of the GC pneumatic control

SV 4: Valve to switch between PTV split and TDU split Figure 4-Pneumatic diagram of the flow of the GC, TDU and PTV during desorption step and injection step

[3]

During thermal desorption step, the TDU is in splitless mode and SV4 valve is closed in TDU. The SV3

valve needs to be open, so there is no overflow in the column.

During sample injection, the PTV is in splitless mode, SV3 valve is closed in PTV. After the splitless

mode time the SV3 valve is open again. [3]

Mode B (splitless desorption, split injection):

Mode B is used whenever circumstances necessitate that not all the adsorbed material on the TDU

needs to reach the column.

Step 1: Every component, desorbed from the tenax in the TDU, will be carried by helium gas and

trapped on the tenax in the cold PTV.

Step 2: The components present on the tenax in the PTV, are splitted. This means that only a small

part reaches the column. The other part is vented through the split vent. [3]

During the thermal desorption step, the TDU is in splitless mode and the SV 4 valve is closed in TDU.

The SV3 valve needs to be open, so there is no overflow in the column (see figure 4, desorption step)

During the sample injection, the PTV is in split mode, the SV 3 valve is open in PTV. This SV 3 valve

controls the split ratio. The split ratio is the ratio of the flow in the column to the flow through the

PTV. [3]

4 ml/min

PTV

1 ml/min

7 ml/min

3 ml/min

7

I.1.1.3 Column

After the sample introduction the components reaches the column. This column contains a thin layer

of a non-volatile chemical, called the stationary phase and can be polar or non polar. The non-volatile

chemical can be coated onto the walls of the column or can be coated onto an inert solid that is

added to column. Components in the gas phase are carried through the column by the carrier gas.

They will selectively interact with the stationary phase of the column. The strength of interaction

depends on the polarity of the stationary phase and the polarity of the component. If the component

is a polar component, it will interact strongly with a polar stationary phase. The column is heated

with a temperature program. Because of its stability and the possibility of decomposition of its

components, the temperature of the column has a limit.

The column used in Scentarom is CP-Sil 8 CB. CP-Sil 8 CB is a non polar column. CP-Sil 8 CB has a

stationary phase of dimethyl polysiloxane with 5 % of the dimethyl replaced with diphenyl. Dimethyl

polysiloxane is a non polar component, but by replacing 5% of dimethyl in diphenyl the column will

be slightly less non polar, but still very non polar. Scentarom also uses carbowax as column.

Carbowax is a more polar column than CP-Sil 8 CB. In this work only CP-Sil 8 CB will be used. [1, 2]

I.1.1.4 Detector

There are several detectors used in gas chromatography. The most common used detectors are

flame ionisation detector (FID) and thermal conductivity detector (TCD).

When the GC is combined with a mass spectrometer, the GC separates the components and the mass

spectrometer serves as a detector.

I.1.2 The GC/MS Interface When the GC is combined with a MS, an interface is required. The interface transports the effluent

from the gas chromatograph to the mass spectrometer. It is a small tube on a high temperature

about 150°C. The temperature of the interface must be programmed at a certain temperature , so

the components neither condense in the interface nor decompose before entering the MS. [1, 7]

I.1.3 MS The mass spectrometer measures the mass-to-charge ratio (m/z) of gas phase ions and provides a

measure of the abundance of each ionic species. Components that are separated by the GC go in the

MS. In the MS, the components will be ionized through electron impact (bombardment). The charged

ions will be placed in a modulated electromagnetic field and sorted by their mass-to-charge ratio in

high vacuum. [7]

I.1.3.1 Sample inlet

In stand alone MS, the entire process from ionisation to detection takes place in gas phase and in

vacuum.

In sample inlet, components go to the gas phase. Liquids and solids will go in a gas chamber to

proceed in gas phase. Volatile components will evaporate immediately by vacuum. This is called

“cold inlet system”. Non volatile components will go in gas phase by heating. This is called “heated

inlet system”.

When using the hyphenated technique GC/MS, components leaving the column are already in gas

phase. Only a small quantity will reach the ion source. Carrier gas will be eliminated. [7]

8

I.1.3.2 Ionization system

The most common used ionization technique is the electron ionization (EI) (figure 5). Gaseous

molecules are brought in vacuum in the ionization chamber by column flow. In this chamber a beam

of electrons are produced by a heated filament and molecules will be ionized and defragmented by

the electrons. Resulting ions are then accelerated by the repeller and brought to the mass analyzer.

The repeller is positively charged compared to this ionization chamber. [7, 8]

Figure 5-Schematic figuration of electron ionization

[9]

The beam of electrons are perpendicular to the beam of molecules, so electrons clash with

molecules. Electrons that will not clash with the molecules will be collected on the trap. When

clashing a molecule, an electron is removed from the molecule and produces a positively charged ion

corresponding to the relative molecular mass of the sample being analysed. Also the molecule will

disintegrates in different pieces. Every piece is typical for the molecule, thus the mass spectrum gives

a fingerprint of the molecule. [8]

I.1.3.3 Mass analyzer

As mass analyzer a quadrupole analyzer (figure 6) is the most common used. A quadrupole analyzer

consists of four parallel rods that have fixed DC and alternating RF potentials applied to them. Ions

produced from the EI are focussed and passed along the middle of the 4 rods of the quadrupole

analyzer. The motion of the ions depends on the electric fields. So only ions with a particular m/z will

have a stable trajectory and will reach the detector, corresponding to a specific RF potential. Other

ions will disappear among the rods of the quadrupole analyzer. To bring ions of different m/z into

focus on the detector the RF is varied and in this way a mass spectrum is built up. The quadrupole

analyzer is used in hyphenated techniques, because of it high scan speed: it can measure up to 500

mass units in 0,005s. In Scentarom the speed of the mass analyzer is up to 4,45 scans per second. 1

scan consists of a mass spectrum of an m/z of 35 to 350. [7, 10]

9

Figure 6-Schematic figuration of a quadrupole analyzer

[11]

I.1.3.4 Ion detector

The most common used ion detector is the electron multiplier (figure 7). It will generate an increased

signal through which the ions can be detected. The electron multiplier consists of a series of dynodes

and is operating in a vacuum. When the ions reach the first dynode, the dynode will emits secondary

electrons. These generated electrons are multiplied in a cascade by the following dynodes. At the

end of the dynodes a cluster of secondary electrons will reach the anode that measures the signal.

The amplified signal will lead to the computer coupled at the MS and GC. [12]

Figure 7-Scheme of an electron multiplier

[13]

I.1.4 Computer The computer is connected to the MS by a converter which converts the signal of the MS in a signal

that can be registered by the computer. The signal is expressed into values of mass versus

abundance. The computer also registers the chromatogram of the GC.

Furthermore, the computer contains a data base of all possible mass spectra of many different

components. It compares the spectrum of the MS with the spectra in the data base. When there is a

match, the computer can tell which component it is.

In the computer there is also a software for creating several methods and adjusting parameters for

obtaining required results.

10

I.2 Sample preparation Sample preparation is needed whenever a sample contains analytes present in a complex matrix. In

this case the sample cannot be used as such and preliminary steps must be taken to prepare the

sample so it is fitted to the instrumental analyses to which it is to be subjected.

During sample preparation there is an enhanced risk of contamination and especially for SHS and

DHS, as sensibility increased considerably. Contamination can often become a major problem.

A few sample preparation techniques will here be described briefly.

I.2.1 Static headspace The word “headspace” here refers to the gas phase located above a liquid or solid phase present in a

sealed vial. The partition of volatile organic components (VOC’s) into the gas phase depends on

several factors a.o. the solubility in water or solvent (hydrophilic or hydrophobic), polarity, ionic

nature of analyte and solvent, molecular weight and temperature.

A liquid and/or solid sample is placed in a sealed vial. The sealed vial has an inert septum, mostly as a

result of teflon coating, and thus should be inert for adsorption of volatile components.

In static headspace an equilibrium is reached between the sample and gas phase. The gas phase can

be partially removed with a syringe and injected in the GC.

Another method for removing the gas phase is, placing an adsorbent material in a sealed vial so

volatile components will adsorb on the adsorbent material. The adsorbent material with the trapped

volatile components will be placed in a TDU. [14, 15]

I.2.2 Dynamic headspace

I.2.2.1 Description

Dynamic headspace (DHS) is a dynamic extraction technique. In DHS, VOC’s are continuously swept

away from the headspace onto a trap by a flow of inert carrier gas. An equilibrium is never reached

between the gas phase and the sample. The inert carrier gas can be nitrogen or helium. Sometimes

the inert carrier gas can be air, but there is a risk for oxidation. To release the components from the

trap, rapid heating is the most efficient method when using an adsorption material. Releasing the

components from the trap can be done in the TDU (see chapter I.1.1.2 Sample introduction, page 3).

The trap used in experimental work is tenax TA. [14, 16]

In practice, the DHS device contains 2 units (figure 8): a DHS station and a DHS carriage. The sample

is placed in a vial in the DHS station while the DHS carriage holds the tube containing the tenax TA.

This tube shall later be placed in the TDU unit for desorption. The DHS carriage can move forward, so

it can be placed above the sample in the DHS station.

DHS can be used for analysis of flavours and fragrances.

11

Figure 8-DHS device

[17]

I.2.2.2 Trapping of components

Figure 9-Schematic overview when trapping all components

[18]

Figure 9 gives an overview of the several steps of DHS when trapping all components. This includes

also all components which are already in the headspace above the sample before the DHS run starts.

Step 1: Before the analysis

The glass vial with the sample is sealed with a septum. Some analytes are already in the headspace

above the sample (gas phase).

Step 2: Incubation

The glass vial is placed into the DHS station. During incubation step the sample is heated up. The

incubation step includes also an agitation step, so the sample can be agitated if desired.

Step 3: Trapping

A TDU tube with an adsorption material is placed into DHS carriage above the glass vial. A double

needle is pushed through the septum into the glass vial, 1 needle for the inlet of the carrier gas in the

glass vial and 1 needle for the outlet of the carrier gas with the analytes. The carrier gas flows over

the sample and transfers the components from the headspace onto the adsorption material in the

tube above it. The carrier gas used is mostly the same as the carrier gas that will be used in the

subsequent GC analysis. During trapping the components the volume and the flow of the carrier gas

can be selected in the software.

DHS carriage

DHS station

12

During trapping 3 parameters are linked to each other:

Trapping gas volume (V)

Trapping gas flow (F)

Trapping time

Only 2 of the 3 parameters can be adjusted. Here the trapping gas volume (V) and the trapping gas

flow (F) can be adjusted. By adjusting the trapping gas volume at a constant gas flow, the trapping

time will change.

Step 4: Drying (optional)

For removing water or solvent, drying is used. Drying is done in a empty glass vial (the “drying vial”).

A defined gas flow is transferred through the drying vial and over the adsorption material in the tube.

This step is optional.

Step 5: Desorption and GC analysis

Adsorption material with the analytes, is transferred to the TDU and here the desorption and

injection step will start (see chapter I.1.1.2.2 PTV combined with TDU, page 3). [18]

In this work a DHS analysis is performed in 2 steps: incubation step (incl. agitation step) and trapping

step. Every step has a specific time and temperature.

Every step has several parameters that can be adjusted. These parameters will be described in the

experimental part of this work.

Time

Incubation step

Agitation step

Trapping step

Δti1 Δti2

Δti Δttr

Tsample i Ttr Temperature Tsample tr

13

Figure 10 gives an overview of the GC/MS (Agilent) used in Scentarom. The MPS (Gerstel) is the robot

that prepares and injects the samples. Dynamic headspace is a fully automated technique through

the use of the MPS robot.

1. Gas chromatograph

2. Mass spectrometer

3. Dynamic headspace (DHS)

4. Thermal desorption unit (TDU)

5. MPS robot

6. Tray TDU-tube Figure 10-GC/MS with configuration for DHS analysis

[19]

4 3

1 2

5

6

14

I.3 Flow charts

Figure 11-Schematic overview of GC/MS and sample preparation

[15]

I.1.1 Gas chromatography (GC)

Injection technique

Injector types

Split/splitless

(Cold) On-column

PTV Columns

I.1.2 Mass spectrometry (MS)

I.2. Sample preparation

Off-line

Filtration

Extraction

Soxhlet

Liquid-liquid

SPE Distillation

...

On-line

Headspace (HS)

Dynamic headspace (DHS)

Purge & trap

"Sweep"

Thermal extraction (TE)

Static headspace (SHS)

"Classical"

Using traps

SPME

Pyrolysis

...

15

Figure 12-Schematic overview of trapping and releasing

[15]

Trapping

Methods

Adsorption

Absorption

Materials

Zeolit

Tenax

Carbo(Trap)

PDMS

Releasing Desorption

Thermal (TDU)

Solvent

16

II Experimental part

The following subjects will be discussed:

Short overview of the several experimental methods used

Study and description of contamination caused during DHS analysis

Parameters description and adjustment for optimal analytical results

Short comparison between DHS and previous SHS techniques used in Scentarom

Analysis of odorants in a shampoo using DHS technique

II.1 Short experimental method descriptions The following methods are used in this experimental part and will be explained:

Blank TDU

Blank DHS

DHS analysis of sample

Figure 13 gives an overview of the several methods. There are 10 time laps described in the figure.

1. Incubation step DHS

During the incubation step the sample vial will be heated up and agitated if desired.

2. Trapping step DHS

During trapping step the components, present in the test sample, will be trapped on the

tenax in the DHS carriage.

3. Transport of TDU-tube from DHS to TDU

4. Pressurize

The pressurize time lap is the time the GC/MS needs to set all the parameters at start

position.

5. TDU heating

During TDU heating the TDU will heat up at a constant rate.

6. TDU hold time

The TDU hold time is the time the TDU stays on it highest temperature.

Time lap 4 and 5 are the desorption step (see chapter I.1.1.2.2 PTV combined with TDU, page

3).

7. TDU cooling

During TDU cooling the TDU cools down.

8. PTV heating + start GC runtime

During the PTV heating, the PTV will heat up with a constant speed.

The GC runtime starts with 1 minute solvent delay. This means that the detector starts to

collect the data after 1 minute. During solvent delay the solvent will reach the detector.

9. PTV hold time

PTV hold time is the time the PTV stays on it highest temperature.

Time lap 8 and 9 are the injection step (see chapter I.1.1.2.2 PTV combined with TDU, page

3).

10. Running

17

II.1.1 Blank TDU The method blank TDU consists of 7 time laps (figure 13):

During blank TDU time lap 1,2 and 3 are not performed. The GC starts directly with pressurize.

4. Pressurize

The duration of this time lap is 1 min. The temperature of the TDU will be 50 °C, the

temperature of the PTV will be 20 °C, the GC-oven will have a temperature of 50 °C and the

flow will be 24 ml/min.

5. TDU heating

The duration of the TDU heating is 2 min. The TDU will heat up with a velocity of 100 °C/min

and the flow will be 54 ml/min.

6. TDU hold time

This is the time lap the TDU stays on a temperature of 250 °C and a flow over the TDU of 54

ml/min. The duration of this time lap is 5 min.

7. TDU cooling

The TDU will cool down to 90 °C in 15 s. The flow is 54 ml/min.


Here the PTV will heat up with a speed of 10 °C/s for 25 s. The flow over the PTV will be 14

ml/min. The runtime of the GC starts and after 1 minute solvent delay the detector will

collect the data of the analysis.

9. PTV hold time

This is the time lap the PTV stays on a temperature of 250 °C. The duration of this time lap is

10 min. The flow goes from 14 ml/min to 24 ml/min.

10. Running

The GC will run for 23 min 40 s with a velocity of 10 °C/min until a temperature of 240 °C is

reached. This is the green line of GC-oven in figure 13.

Data are collected as a control for the blank-level of the blank TDU.

II.1.2 Blank DHS The method blank DHS consists of 10 time laps (figure 13):


During the incubation step the transfer heater will be 150 °C, the incubation temperature

30°C, the agitator speed 500 rpm. The duration of the incubation step is 1 min.


During the trapping step the transfer heater will be 150 °C, the incubation temperature 30 °C

and the trapping flow 50 ml/min. The duration of the incubation step is 2 min.

3. Transport of TDU-tube from DHS to TDU

During the transport the DHS flow will be 1 ml/min.

4. Pressurize




5. TDU heating



18

6. TDU hold time



7. TDU cooling




ml/min.

9. PTV hold time



10. Running


reached. This is the green line of GC-oven in figure 13.

Data are collected as a control for the blank-level of the blank DHS.

II.1.3 DHS analysis of sample The method DHS analysis of sample consists of 9 time laps (figure 13):


During the incubation step the transfer heater will be 150 °C, the incubation temperature

30°C, the agitator speed 500 rpm. The duration of the incubation step is 1 min.


During the trapping step the transfer heater will be 150 °C, the incubation temperature 30 °C

and the trapping flow 50 ml/min. The duration of the incubation step is 2 min.

3. Transport of TDU-tube from TDU to DHS

During the transport the DHS flow will be 1 ml/min.

4. Pressurize




5. TDU heating



6. TDU hold time



7. TDU cooling




ml/min.

9. PTV hold time



19

10. Running


reached. This is the red line of GC-oven in figure 13.

1 2 3 4 5 6 7 8

9 10

25 °C

150 °C

1 min 0 min 3 min 4 min 5 min 7 min 12 min 15 s

24 s 0 s 1 min 10 min 25 s 23 min 40 s 65 min 20 s 74 min 20 s

30 °C

500 rpm

50 ml/min

5 ml/min 1 ml/min

5 ml/min

30 °C

300 °C

50 °C 100 °C/min

250 °C 90 °C 123 °C

20 °C 10 °C/s

250 °C 100 °C

50 °C 3 °C/min

250 °C

Solvent delay

24 ml/min

50 ml/min

10 ml/min 24 ml/min

10 °C/min

250 °C

SS SS SS SS

DHS Trap

Transfer

heater

Incubation

temperature

(Tsample i)

Agitator speed

Trapping flow

Incubation

temperature

(Tsample tr)

TDU transfer

TDU

temperature

PTV

temperature

GC

temperature

Data

Flow

GC/MS

Figure 13-Schematic overview of parameters of DHS and GC/MS Sample preparation GC/MS analysis

SS

12 min

20

DHS

21

TDU head

(inox)

O-rings

(rubber)

Tenax tube

(glass)

Vial

(glass)

II.2 Contamination

II.2.1 Introduction Scentarom produces flavours and fragrances for various applications. As a consequence a strong

background odour is present in the laboratories and production facilities. Thus, whenever the

adsorbent material (tenax) is in contact with the open environment within Scentarom, contamination

inevitably takes place. Detailed study of this contamination is absolutely necessary in order to master

(and if possible to eliminate) its influence on later analytical results.

II.2.2 Sources where contamination can occur To understand the sources of contamination next chapters has to be read first:

chapter I.1.1.2.2 PTV combined with TDU, page 3

chapter I.2.2 Dynamic headspace, page 10

PTV-liner TDU-tube sample vial Figure 14-Figuration of PTV-liner, TDU-tube and sample vial

Figure 14 gives a figuration of PTV-tube, TDU-tube and sample vial used in GC/MS. For photos of PTV-

tube, TDU-tube and sample vial: see attachment 2, page 55.

The following steps can cause contamination:

Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax liner)

TDU-tube in TDU-tray

PTV-liner in PTV

TDU-tube in TDU

Unlocking of TDU

Transporting TDU-tube to DHS carriage

Septum of sample vial

DHS carriage

DHS station

Sealing

(graphite)

Tenax

Tenax liner

(glass)

Tenax

Septum

(rubber +

teflon)

22

O-rings on TDU head

Carriers

II.2.3 Measurement of contamination

II.2.3.1 Preservation conditions of adsorption material (tenax in tenax tube and tenax in

tenax liner)

There is a strong background odour present within Scentarom. This background odour causes a lot of

contamination when the tenax in tenax tube and tenax liner are in contact with this background

odour. To reduce the contamination of the tenax in tenax tube and tenax liner a serie of blank TDU

has be performed before starting a DHS analysis.

Former test shows that the contamination can be reduced by keeping the tenax in a glass jar sealed

with aluminium foil. There will be less contamination, so less blank TDU will have to be performed to

reduce the contamination. (see thesis Pien and chapter II.2.4 Conclusion, page 30)

II.2.3.2 TDU-tube in TDU-tray

The TDU-tube needs to be placed in the TDU-tray when starting a DHS analysis. When performing a

DHS analysis the TDU-tube goes from TDU-tray to the DHS station. The time the TDU-tube is in TDU-

tray is 5 min. The contamination of the TDU-tube in TDU-tray is given in figure 15.

Figure 15-Chromatogram of contamination of TDU-tube (containing tenax) in tray

Conclusion:

TDU-tube in TDU-tray causes contamination. This contamination can be eliminated by performing

analyses in a sequence. When performing a sequence of analyses, the movement of the TDU is done

by the MPS robot. This robot moves the tenax tube in the TDU directly to the DHS station without

staying in the tray. The several steps of the MPS robot when performing a sequence of

measurements are given in attachment 1, page 54.

II.2.3.3 PTV-liner in PTV

When there is no analysis performed the PTV-liner stays in the PTV for technical and practical

reasons. The PTV is not airtight, so during standby the PTV-liner is exposed to the open environment

23

within Scentarom. This contamination is tested when the PTV-liner was respectively 1 day, 3 days

and 1 week present in the PTV. The results are given in figure 16.

Figure 16-Results of PTV-liner in PTV

The chromatograms of these tests are given in attachment (see attachment 3, page 55-56).

Conclusion:

The PTV-liner causes contamination. When no analyses are performed, the tenax in the tenax liner

adsorbs odour molecules present in the open environment within Scentarom. The tenax liner needs

to be free of contamination before starting a DHS analysis. To reduce the contamination of the PTV-

liner in the PTV a sequence of blank analyses (i.e. TDU blank) needs to be done. After a few analyses

the tenax in the PTV-liner will be blank.

In all further tests the PTV-liner will remain in the PTV during each analysis.

II.2.3.4 TDU-tube in TDU

The TDU-tube is kept outside Scentarom to reduce the contamination of the tenax in the tenax tube.

The contamination is tested when the TDU-tube was 3 days, 1 week and 2 months exposed in an

open environment outside Scentarom. The results are given in figure 17.

0,00E+00

1,00E+08

2,00E+08

3,00E+08

4,00E+08

5,00E+08

6,00E+08

7,00E+08

8,00E+08

9,00E+08

1,00E+09

Are

a (a

bso

lute

)

Total area of contamination of PTV-liner in PTV

1 day

3 days

1 week

24

Figure 17-Results of contamination of TDU-tube in TDU

The chromatograms of these analysis are given in attachment (see attachment 4, page 56-57).

Conclusion:

The tenax in the tenax tube adsorbs odour molecules even when the tenax is not exposed to the

open environment within Scentarom. To eliminate this contamination the tenax in the tenax tube

needs to be free of contamination before starting a DHS analysis. To reduce the contamination of the

TDU-tube in TDU, a sequence of blank TDU needs to be done. After a few analyses the tenax in the

TDU-tube will be blank.

To reduce the total time of measurements, the TDU-tube and PTV-liner will be made free of

contamination during the same sample analysis sequence.

II.2.3.5 Unlocking of TDU

When the TDU-tube goes to the DHS carriage, the TDU will be unlocked and open for 45 seconds. The

contamination of this step is tested and the result is given in figure 18.

0,00E+00

5,00E+07

1,00E+08

1,50E+08

2,00E+08

2,50E+08

3,00E+08

3,50E+08

4,00E+08

4,50E+08

Are

a (a

bso

lute

) Total area of contamination of TDU-tube in TDU

3 days

1 week

2 months

25

Figure 18-Chromatogram of contamination when the TDU is open for 45 seconds

Conclusion:

When the TDU is unlocked and open for 45 seconds, it causes negligible contamination.

II.2.3.6 Transporting TDU-tube to DHS carriage

When the TDU-tube is transported to DHS carriage, the TDU-tube will be exposed to the open

environment within Scentarom for 1 minute. The contamination of this step is tested and the result is

given in figure 19.

Figure 19-Chromatogram of test of transporting TDU-tube to DHS carriage

Conclusion:

Transporting the TDU-tube to the DHS carriage will not cause much contamination, so this step has

also negligible effect on the presence of contamination.

26

II.2.3.7 Septum of the sample vial

Figure 20-Figuration of septum of sample vial

The sample vial septum contains rubber and teflon (figure 20). Rubber is known for its adsorption of

odour molecules. In this test an empty sample vial with the same septum is measured for several

consecutive times. The result is given in figure 21.

Figure 21-Result for contamination of septum of the sample vial

Conclusion:

The contamination increases slightly. This can be explained by the fact that when the rubber and

teflon are pierced, the contamination of the rubber (odorous material adsorbed on the rubber) can

0,00E+00

1,00E+07

2,00E+07

3,00E+07

4,00E+07

5,00E+07

6,00E+07

7,00E+07

8,00E+07

1 2 3 4 5 6 7 8 9 10 11 12 13

Are

a (a

bso

lute

)

Total area of contamination of different blank DHS analyses

rubber

teflon

Movement of needles in DHS carriage

Needles

27

go into the sample vial through the pierced teflon. The described contamination can be avoided

through the use of new septa for each analysis. Changing the septa for every analysis is however not

expected to be essential.

II.2.3.8 DHS station and DHS carriage

The DHS station and DHS carriage are not airtight. There are o-rings made of rubber present in the

DHS carriage, these rubber o-rings can also cause contamination. The o-rings cannot be removed for

technical reasons, so the contamination caused by the o-rings in the DHS carriage cannot be reduced.

The contamination of the DHS station and DHS carriage is tested and the results are given in figure

22.

Figure 22-Results for contamination of blank DHS

The chromatogram of blank DHS is given in attachment (see attachment 5, page 57).

Conclusion:

The DHS station and carriage causes a lot of contamination. The amount of contamination is shown

by the difference in total area in figure 22. This significant contamination cannot be reduced.

However the influence of the contamination on the analytical results can be minimized by increasing

the split ratio. (see chapter II.3.3 Split ratio R in PTV, page 33).

II.2.3.9 O-rings on TDU-head

The O-rings of the TDU-head are made of rubber. Rubber is known for its adsorption of odour

molecules. The contamination is tested when the TDU head is exposed in the open environment

within Scentarom for respectively 1 min and 1 day. It is also tested when the TDU head is wrapped in

0,00E+00

5,00E+06

1,00E+07

1,50E+07

2,00E+07

2,50E+07

3,00E+07

3,50E+07

Are

a (a

bso

lute

)

Total area

Transporting TDU-tubeto DHS carriage

Blank DHS

28

aluminium foil and exposed in the open environment within Scentarom for respectively 1 day and 1

week. The results are given in figure 23.

Figure 23-Contamination of O-rings on TDU head

The chromatograms of these tests are in attachment (see attachment 6, pages 57-58).

Conclusion:

The O-rings on the TDU head causes contamination. To avoid this contamination the TDU heads are

kept in an open environment outside Scentarom.

II.2.3.10 Carrier

When preparing a test sample for DHS analysis, the sample needs to be diluted. Silica and a

carbohydrate are used as carrier. Silica and carbohydrate are solids that can adsorb odour molecules,

so can cause contamination. The contamination of carbohydrate and silica is tested when exposed in

open environment within Scentarom and when exposed in an open environment outside Scentarom.

The results are given in figure 24.

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

Are

a (a

bso

lute

)

Total area of O-rings on TDU head

1 min

1 day

1 day in aluminium foil

1 week in aluminium foil

29

Figure 24-Results of contamination of carbohydrate and silica

The chromatograms of these test are in attachment (see attachment 7, page 59-60).

Conclusion:

Carbohydrate causes a lot of contamination. Also silica adsorbs components present in the open

environment within Scentarom. To reduce this contamination carbohydrate and silica need to be

kept in a glass jar in an open environment outside Scentarom. After each use, the carriers need to be

exposed to open air outside Scentarom during several hours before closing the jar.

0,00E+00

5,00E+07

1,00E+08

1,50E+08

2,00E+08

2,50E+08

Are

a (a

bso

lute

) Total area of carbohydrate and silica

Carbohydrate exposedin open environmentat Scentarom

Carbohydrate 2 daysexposed in openenvironment outsideScentarom

Silica exposed in openenvironment atScentarom

Silica 2 days exposed inopen environmentoutside Scentarom

30

II.2.4 Conclusion To reduce the contamination of the TDU-tube in TDU and the PTV-liner in PTV a sequence of blank

analyses needs to be done. The results of these blank analyses are given in figure 25.

Figure 25-Chromatograms of blank TDU

The first chromatogram in figure 25 is the contamination of the tenax before performing a blank TDU

and the second chromatogram is the contamination the tenax after performing a blank TDU.

After 2 measurements the tenax in the tenax tube and tenax liner are reduced to almost blank.

These analyses must be done before performing a DHS analysis.

Also the septum of the sample vial needs to replaced after every analysis.

After using carbohydrate and silica these adsorbens needs to be exposed in an open environment

outside Scentarom.

before

after

31

Thus:

When performing a DHS analysis, the following sequence is performed.

1. Blank TDU

Blank TDU is performed to eliminate the contamination on the tenax. This analysis will be

repeated till the tenax is blank. When the tenax is blank (see figure 25), blank DHS will start.

2. Blank DHS

Blank DHS is a DHS analysis with an empty sample vial. This is performed to evaluate the

contamination of a DHS analysis.

3. DHS analysis of sample

This is a DHS analysis of a sample in the sample vial.

32

II.3 Parameters

II.3.1 Introduction The DHS technique can be described in 3 steps: incubation step, trapping step and drying step (see

chapter I.2.2.2 Trapping of components, page 11). Every step of the DHS device has several

parameters that can be adjusted.

The next parameters will be tested:

Split ratio R in PTV (see chapter I.1.1.2.2 PTV combined with TDU, page 3)

Sample temperature (Tsample i and Tsample tr)

Incubation time Δti (Δti = Δti1 + Δi2)

Trapping gas flow F (V=constant)

Trapping gas volume V (F=constant)

The trap temperature (Ttr) is also a parameter that can be tested. This parameter is not tested in this

work. Indeed, the temperature in the laboratories of Scentarom is 25°C, so a lower temperature is

not possible. A higher temperature is has no advantage, because the components with a low boiling

point will not adsorb on the tenax in the tenax tube when trapping the components.

Table 2-Overview of the split ratio and the start split ratio

Range Start

Split ratio (R) 1/1-1/1250 1/10

Table 3-Overview of the parameters of steps of the DHS and the start parameters

Range Start

Transfer Heater 50-150 °C 150°C

Incubation step (incl. agitation step)

Incubation Temperature (Tsample i)

10-200°C 30°C

Total incubation Time (Δti)

0-600 min 1 min

Agitator On Time (Δti2) 0-600 s 60 s

Agitator Off Time 0-300 s 1 s

Agitator Speed 250-1500 rpm 500 rpm

Trapping step Volume (V) 10-100000 ml 100,0 ml

Flow (F) 5-100 ml/min 50,0 ml/min

Trap Temperature (Ttr) 20-70 °C 25 °C

Incubation Temperature (Tsample tr)

10-200 °C 30 °C

Time (Δttr) / 2 min

Drying step (optional)

Volume 10-100000 ml 0 ml

Flow 5-100 ml/min /

Trap Temperature (Tdr)

20-70 °C /

Incubation Temperature (Tsample dr)

10-200 °C /

Time / /

Table 2 gives the split range and start value.

33

Table 3 gives an overview of the several parameters used in the DHS. It also gives the range of the

parameters. All these parameters will be tested, and the best result will be selected.

The last column in table 3 shows the start values of the parameters. These values will be adjusted to

the most efficient value.

II.3.2 Test sample The test sample used for parameter control consists of 12 components (table 4).

Table 4-Overview of components in test sample

Number in chromatogram

(see attachments)

Component (structures see attachment 8,

page 60)

Boiling Point (°C)

Molecular weight (g/mol)

Retention time (min)

1 diacetyl 88 86 5,2

2 ethyl acetate 77 88 5,5

3 cis-3-Hexenol 157 100 11,7

4 benzaldehyde 178 106 16,7

5 ethyl hexanoate 168 128 18,0

6 phenylethyl alcohol 220 122 24,1

7.I & 7.II citronellal 206 154 25,8 + 26,3

8 eugenol 256 164 35,3

9 b-ionone 128 192 40,8

10 g-undecalactone 297 184 44,5

11 thibetolide 280 240 54,4

12 benzyl salicylate 335 228 55,7

Boiling point and molecular weight were found in data sheets of the products.

The elution time of the components depends a.o. on boiling point, polarity and molecular weight of

each components and column characteristics. These components show a good dispersion in the

chromatogram. These components are representative for the most common chemical classes used

when producing fragrance and flavours.

II.3.3 Split ratio R in PTV See chapter I.1.1.2.2 PTV combined with TDU, page 3.

Split ratio is an important parameter.

The concentration of the samples has been adapted so as to keep the amount of all injected

components on the column independent from the split ratio used. After each analysis a blank DHS

was performed to evaluate the contamination (see figure 26)

The parameters of DHS for performing a DHS analysis are in attachment (see attachment 9, page 61).

The following split ratios have been tested (table 5):

Table 5-Overview of the different split ratios and corresponding concentrations that have been tested

Test 1 Test 2 Test 3 Test 4

Split ratio (R) 1/10 1/25 1/50 1/100

Concentration (ppm) 100 250 500 1000

34

Results:

Figure 26-Total area of test sample and total area of contamination

0,00E+00

5,00E+07

1,00E+08

1,50E+08

2,00E+08

2,50E+08

3,00E+08

3,50E+08

4,00E+08

4,50E+08

Splitratio 1/10 Splitratio 1/25 Splitratio 1/50 Splitratio 1/100

are

a (a

bso

lute

)

split ratio

total area

Total area of testsample

Total area of blankDHS

Split ratio 1/10

Split ratio 1/25

35

Figure 27-Chromatogram of blank analysis of split ratio 1/10, 1/25, 1/50 and 1/100

Figure 27 shows the chromatograms of a DHS analysis of an empty glass vial, so the peaks on the

chromatogram are contamination present in the glass vial or contamination present during

performing a DHS analysis.

Conclusion:

By increasing the split ratio, less contamination will reach the column and will be detected. This

means that the contamination is present in the DHS unit and not in the test sample. Increasing the

split ratio from 1/50 to 1/100, has minor advantage. The chosen split ratio for further testing will be

1/50 and concentration of the samples will be 500 ppm.

II.3.4 Sample temperature (Tsample i and Tsample tr) during incubation step and

trapping step See chapter I.2.2.2 Trapping of components, page 11.

The sample temperature is the temperature of the sample in the glass vial during incubation step

(Tsample i) and trapping step (Tsample tr) and are kept equal.

The concentration of the samples will be 500 ppm, the split ratio 1/50. (see attachment 10, page 61).

The following incubation temperatures will be tested (table 6):

Table 6-Overview of the different incubation temperatures that have been tested

Test 1 Test 2 Test 3 Test 4 Test 5

Sample temperature Tsample i and Tsample tr (°C)

30 50 65 75 85

Split ratio 1/50

Split ratio 1/100

36

Results:

Figure 28-Influence of incubation temperature on the relative area and the total area of the components trapped

The chromatograms of these tests are in attachment (see attachment 11, page 62-63)

0

5

10

15

20

25

30

are

a (r

ela

tive

in %

)

Components

Relative area

30°C

50°C

65°C

75°C

85°C

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

3,50E+09

4,00E+09

4,50E+09

are

a (a

bso

lute

)

Total area

30°C

50°C

65°C

75°C

85°C

37

Conclusion:

The sample temperature has a significant influence on the signal. When using a low incubation

temperature the components with a smaller elution time will have a higher area.

When using 30 °C, benzyl salicylate is not detectable. When using 85°C diacetyl and ethylacetate are

almost not detectable. An incubation temperature of 65°C and 75°C shows the best results. An

incubation temperature of 65°C will be selected, as a higher temperature could decompose the

components.

When analysing a sample with components that have a lower retention time, an incubation

temperature of 30 °C will be preferred.

II.3.5 Total incubation time Δti See chapter I.2.2.2 Trapping of components, page 11.

The total time Δti consists of two time intervals: incubation time and agitation time. The total

incubation time Δti and agitation time Δti2 can be selected. By adjusting those 2 parameters the

incubation time Δti1 will change automatically.

The concentration of the sample is 500 ppm, the split ratio is 1/50 and the incubation temperature is

65°C. (see attachment 12, page 64).

The following incubation times will be tested (table 7):

Table 7-Overview of the different incubation times that have been tested

Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7

Total incubation time Δti 0 min 15 s 1 min 2 min 5 min 10 min 15 min

Agitation time Δti2 0 15 s 1 min 2 min 5 min 10 min 10 min

Incubation time Δti1 (min) 0 0 0 0 0 0 5 min

38

Results:

Figure 29-Influence of incubation time on the relative area and the total area of the components trapped

The chromatograms of these tests are in attachment (see attachment 13, page 64-66).

0

5

10

15

20

25

are

a (r

ela

tive

in %

)

Components

Relative area

0 min

15 s

1 min

2 min

5 min

10 min

15 min

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

3,50E+09

are

a (a

bso

lute

)

Total area

0 min

15 s

1 min

2 min

5 min

10 min

15 min

39

When there is no incubation step, the total area of the components is low. An incubation step of 15 s

does not give much improvement. One minute incubation gives a significant increase of the total

area, but further increase gives limited ameliorations (see figure 29).

Conclusion:

The incubation step is necessary to trap the components with a higher elution time. It has a

minimum duration required to trap the components with a higher elution time.

To reduce the total time of a DHS analyse an incubation time of 5 minutes will be selected.

II.3.6 Trapping gas flow F (V=constant) See chapter I.2.2.2 Trapping of components, page 11.

When adjusting the trapping gas flow at a constant trapping gas volume, the trapping time (Δttr) will

change.

The concentration of the sample is 500 ppm, the splitratio is 1/50, the incubation temperature is

65°C and the incubation time is 5 minutes. (see attachment 14, page 67).

The following trapping flows will be tested (table 8):

Table 8-Overview of the different trapping gas flows that have been tested


Trapping gas flow F (ml/min) 5 10 25 50 100

Trapping gas volume V (ml) 100 100 100 100 100

Trapping time Δttr (min) 20 10 4 2 1

40

Results:

Figure 30-Influence of trapping gas flow on the relative area and the total area of the components trapped

The chromatograms of these test are in attachment (see attachment 15, page 67-69)

0

5

10

15

20

25

30

are

a (r

ela

tive

in %

)

Components

Relative area

5 ml/min

10 ml/min

25 ml/min

50 ml/min

100 ml/min

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

are

a (a

bso

lute

)

Total area

5 ml/min

10 ml/min

25 ml/min

50 ml/min

100 ml/min

41

Conclusion:

The trapping gas flow has a minor influence on the amount of the components trapped. However

when using a lower trapping gas flow the trapping time will increase. To reduce the total time of an

DHS analysis to a reasonable time, a flow of 50 ml/min will be selected.

II.3.7 Trapping gas volume V (F=constant) See chapter I.2.2.2 Trapping of components, page 11.

When adjusting the trapping gas volume at a constant trapping gas flow, the trapping time (Δttr) will

change.

The concentration of the sample is 500 ppm, the split ratio is 1/50, the incubation temperature is

65°C, the incubation time is 5 minutes and the trapping flow is 50 ml/min. (see attachment 16, page

69).

The following trapping flows will be tested (table 9):

Table 9-Overview of the different trapping gas volumes that have been tested


Trapping gas volume V (ml) 10 50 100 150 200

Trapping gas flow F (ml/min) 50 50 50 50 50

Trapping time Δttr 12 s 1 min 2 min 3 min 4 min

42

Results:

Figure 31-Influence of trapping gas volume on the relative area and the total area of the components trapped

0

5

10

15

20

25

30

are

a (r

ela

tive

in %

)

Components

Relative area

10 ml

50 ml

100 ml

150 ml

200 ml

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

are

a (a

bso

lute

)

Total area

10 ml

50 ml

100 ml

150 ml

200 ml

43

The chromatograms of these tests are in attachment (see attachment 17, page 70-71)

Conclusion:

The trapping gas flow has a considerable influence. When increasing the trapping gas flow the

components with a higher elution time will have a higher area. The total area of components trapped

changes dramatically with the volume V increased. This change seems to be asymptotic. The

difference between 150 ml and 200 ml becomes minor, so 150 ml as trapping gas volume can be

selected.

II.3.8 Reproducibility The concentration of the sample is 500 ppm, the split ratio is 1/50, the incubation temperature is

65°C, the incubation time is 5 minutes, the trapping flow is 50 ml/min and the trapping volume is 150

ml. (see attachment 18, page 72).

The DHS analysis is repeated seven times. Each time a new test sample was prepared. The results are

given in figure 32.

44

Results:

Figure 32-Reproducibility of DHS on the absolute area of each component and the total area of all components trapped

0,00E+00

1,00E+08

2,00E+08

3,00E+08

4,00E+08

5,00E+08

6,00E+08

7,00E+08

8,00E+08

are

a (a

bso

lute

)

Components

Absolute area

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Test 7

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

3,50E+09

4,00E+09

are

a (a

bso

lute

)

Total area

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Test 7

45

Table 10-Total area of the individual analyses

Total area

Test 1 2794640112

Test 2 3569893897

Test 3 2582285857

Test 4 2682901118

Test 5 2521646021

Test 6 3154605386

Test 7 2769350731

Table 10 gives the total areas of the individual analyses.

Test 2 has a higher total area than the other tests, so the Dixon-Q test and the 4s standard has been

performed to evaluate the result of test 2.

Conclusion:

After performing the Dixon-Q test and the 4s standard there can be concluded that test 2 is no

outliner. The reproducibility of these results are sufficient enough to use this method in further

analyses, as Scentarom will be in the future more interested in relative ratio among constituent of

compositions (see attachment 19, page 72).

II.4 Comparing DHS analysis and SHS analysis A SHS-type analysis has been performed and described by Pien earlier [15]. The concentration of the

test sample when performing a SHS is 100 ppm with a split ratio 1/10.

The concentration of the test sample when performing a DHS is 500 ppm with a split ratio 1/50.

Results are given in Figure 33:

0

5

10

15

20

25

30

are

a (r

ela

tive

in %

)

Components

Relative area

DHS

SHS

46

Figure 33-Comparing DHS with SHS on the absolute, relative and total area of the components trapped

The chromatograms of these tests are in attachment (see attachment 20, page 73).

0,00E+00

1,00E+08

2,00E+08

3,00E+08

4,00E+08

5,00E+08

6,00E+08

7,00E+08

are

a (a

bso

lute

)

Components

Absolute area

DHS

SHS

0,00E+00

5,00E+08

1,00E+09

1,50E+09

2,00E+09

2,50E+09

3,00E+09

are

a (a

bso

lute

)

Total area

DHS

SHS

47

Conclusion:

The sensitivity of DHS and SHS are in the same magnitude for this test sample (fragrance compounds

on carrier).

DHS has a higher sensitivity for the central components like citronellal and phenylethyl alcohol.

DHS however is a fully automated technique through the use of the robot, which gives an advantage

over SHS when several samples must be treated.

II.5 Shampoo When analysing the shampoo four different methods of diluting the shampoo were performed. This

is done to see which method gives the best results.

The split ratio is adapted accordingly to keep the responses within dynamic range.

Four different methods:

1. 100 % shampoo

2. 50 % shampoo + 50 % H2O

3. 50 % shampoo + 50 % silica

4. 25 % shampoo + 25 % H2O + 50 % silica

These 4 test shampoos were analysed with a different split ratio and the results are given in Fout!

Verwijzingsbron niet gevonden.:

1. Analysed with split ratio 1/250




100 % shampoo

48

50 % shampoo + 50 % H2O

50 % shampoo + 50 % silica

49

Figure 34-Chromatograms of shampoo with concentration of 100 % shampoo, 50 % shampoo 50 % H2O, 50 % shampoo 50 % silica and 25 % shampoo 25 % H2O 50 % silica

Conclusion:

When analysing the shampoo diluted in silica, all peaks are separated and have symmetrical shape.

When analysing the shampoo with a concentration of 100 % or diluted in 50 % H2O, not all peaks are

separated or have a symmetrical shape, see figure 34 chromatogram one and two. The reason why

these peaks are not separated is not known. Further tests has to be done.

25 % shampoo + 25 % H2O + 50 % silica

50

Conclusion

Study of the DHS technique for fragrant compositions in Scentarom reveals following features:

A. Contamination

Contamination is caused by following steps (+ appropriate reduction):

Preservation conditions of adsorption material (tenax in tenax tube and tenax in tenax

liner)

These materials are now kept in a glass jar sealed with aluminium outside Scentarom.

TDU-tube in TDU-tray

This contamination is eliminated by performing analyses in sequence, so the TDU-tube

doesn’t have to stay in the TDU-tray.

PTV-liner in PTV and TDU-tube in TDU

The PTV-liner and TDU-tube cause contamination when no analyses are performed. This

contamination is eliminated by performing a sequence of blank TDU.

DHS station and DHS carriage

This contamination cannot be reduced. However the influence of the contamination on

the analytical results can be minimized by increasing the split ratio.

O-rings on TDU-head

This contamination will be avoided by keeping the O-rings in an open environment

outside Scentarom.

Carrier

To reduce this contamination the carriers are kept in a glass jar in an open environment

outside Scentarom.

Steps that give negligible contamination:

Unlocking of TDU

Transporting TDU-tube to DHS carriage

Septum of the sample vial

B. The following experimental parameters were tested:

Split ratio R in PTV

Sample temperature (Tsample i and Tsample tr) during incubation step and trapping step

By increasing the sample temperature the components with a higher elution time will

have a higher area.

Total incubation time Δti

The incubation time is necessary to trap the components with a higher elution time. It

has a minimum duration required to trap the components with a higher elution time.

Trapping gas flow F

The trapping gas flow has a minor influence on the amount of the components trapped.

Trapping gas volume V

The trapping gas flow has a considerable influence. When increasing the trapping flow

the components with a higher elution time will have a higher area.

51

As a result, optimal experimental parameter settings are:

Spit ratio: 1/50

Sample temperature: 65 °C

Total incubation time: 5 min

Trapping gas flow: 50 ml/min

Trapping gas volume: 150 ml

C. Comparing DHS analysis to SHS-type analysis:

The sensitivity of DHS and SHS-type are similar for this test sample and test conditions.

However, DHS has a higher sensitivity for the medium volatile components. Furthermore,

DHS is a fully automated technique through the use of the robot, which makes it the method

of preference.

52

Bibliography

1. Fulton G. Kitson, B.S.L., Charles N. McEwen, gas chromatography and mass spectrometry, a practical guide. 1996, California: Academic Press, Inc.

2. Kyaw Thet, N.W. gas chromatography. 2007 [cited 2014 14 february]; Available from: http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Gas_Chromatography.

3. Gerstel, Thermal Desorption Unit TDU with C506-Operation Manual. 2009: Gerstel. 4. Gerstel. Thermal Desorption Unit TDU-certified supplies. 2013 [cited 2014 18 march];

Available from: http://www.gerstel.com/pdf/s00135-702-02-Supplies-TDU-en.pdf. 5. R.I.C., SHS-DHS-TD-TE-SBSE-HSSE. 2012. 6. Lagard, D., G.A. Reiner, and G.W. Christensen. Analysis of the headspace proximate a

substrate surface containing fragrance-containing microcapsules. 2004 [cited 2014 6 may]; Available from: http://www.google.com/patents/US7531365.

7. Verberckmoes, A., Spectroscopische technieken. 2012, Gent. 8. Electron impact. [cited 2014 14 march]; Available from:

http://www.birmingham.ac.uk/facilities/chemical-analysis/mass-spec/electron-impact.aspx. 9. Mass Spectrometry Tutorial. [cited 2014 18 march]; Available from:

http://tera.chem.ut.ee/~jpenchuk/documents/kursused/Mass-spec/2.htm. 10. Gates, P. Quadruple & Triple Quadrupole (QQQ) Mass Analysis. 2009 [cited 2014 14 march];

Available from: http://www.chm.bris.ac.uk/ms/theory/quad-massspec.html. 11. GC/MS-TOF Description. [cited 2014 14 march]; Available from:

http://chromservis.cz/category/gc-ms-tof. 12. Hamamatsu, Electron multiplier tubes and ion detectors, in Photomultiplier Tubes: Basics and

Applications. 2007. p. 12. 13. How electron multipliers work. 2013 [cited 2014 11 march]; Available from:

http://www.sge.com/products/electron-multipliers/how-electron-multipliers-work. 14. Kevin Goodner, R.R., Practical analysis of flavor and fragrance materials. 2011: Whiley. 15. Pien, N., Static headspace characterisation of odorants in complex matrices by hyphenated

GC-MS techniques, in Hogeschool Gent. 2013: Gent. 16. Hübschmann, H.-J., Handbook of GC/MS: Fundamentals and Applications. 2009, Weinheim:

Whiley-VCH. 17. Gerstel. gerstel dynamic headspace DHS. [cited 2014 15 march]; Available from:

http://www.davinci-ls.com/systems/sample-introduction-preparation-systems/69-gerstel-dynamic-headspace-dhs.

18. Gerstel, Dynamic Headspace DHS with TDU and MPS-operation manual. 2014. 19. EVISA. Gerstel GmbH & Co.KG - MulitPurpose Sampler MPS for GC and GC/MS. 2010 [cited

2014 27 february]; Available from: http://www.speciation.net/Database/Instruments/GERSTEL-GmbH-amp-CoKG/MultiPurpose-Sampler-MPS-for-GC-and-GCMS-.

http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Gas_Chromatography

http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Gas_Chromatography

http://www.gerstel.com/pdf/s00135-702-02-Supplies-TDU-en.pdf

http://www.google.com/patents/US7531365

http://www.birmingham.ac.uk/facilities/chemical-analysis/mass-spec/electron-impact.aspx

http://tera.chem.ut.ee/~jpenchuk/documents/kursused/Mass-spec/2.htm

http://www.chm.bris.ac.uk/ms/theory/quad-massspec.html

http://chromservis.cz/category/gc-ms-tof

http://www.sge.com/products/electron-multipliers/how-electron-multipliers-work

http://www.davinci-ls.com/systems/sample-introduction-preparation-systems/69-gerstel-dynamic-headspace-dhs

http://www.davinci-ls.com/systems/sample-introduction-preparation-systems/69-gerstel-dynamic-headspace-dhs

http://www.speciation.net/Database/Instruments/GERSTEL-GmbH-amp-CoKG/MultiPurpose-Sampler-MPS-for-GC-and-GCMS-

http://www.speciation.net/Database/Instruments/GERSTEL-GmbH-amp-CoKG/MultiPurpose-Sampler-MPS-for-GC-and-GCMS-

53

Attachments

Attachment 1-The several steps of the MPS for performing a DHS analysis ........................................ 54

Attachment 2-Figuration of PTV-tube, TDU-tube and sample vial ....................................................... 55

Attachment 3-Chromatograms of contamination of PTV-liner in PTV .................................................. 55

Attachment 4-Chromatograms of contamination of TDU-tube in TDU ................................................ 56

Attachment 5-Chromatogram of blank DHS ......................................................................................... 57

Attachment 6-Chromatograms of contamination of O-ring ................................................................. 57

Attachment 7-Chromatograms of contamination of carrier ................................................................. 59

Attachment 8-Overview of structures of different components .......................................................... 60

Attachment 9-Parameters of DHS for adjusting split ratio ................................................................... 61

Attachment 10-Parameters of DHS for adjusting incubation temperature .......................................... 61

Attachment 11-Chromatograms of analyses for adjusting incubation temperature ........................... 62

Attachment 12-Parameters of DHS for adjusting incubation time ....................................................... 64

Attachment 13-Chromatograms of analyses for adjusting incubation time ......................................... 64

Attachment 14-Parameters of DHS for adjusting trapping gas flow ..................................................... 67

Attachment 15-Chromatograms of analyses for adjusting trapping gas flow ...................................... 67

Attachment 16-Parameters of DHS for adjusting trapping gas volume ................................................ 69

Attachment 17-Chromatograms of analyses for adjusting trapping gas volume ................................. 70

Attachment 18-Parameters of DHS when performing a DHS ............................................................... 72

Attachment 19- Reproducibility of DHS on the relative area of each component trapped ................. 72

Attachment 20-Chromatograms of DHS and SHS comparison ............................................................. 73

54

Attachment 1-The several steps of the MPS for performing a DHS analysis

The dynamic headspace is a fully automated technique. It is automated by a robot above the GC/MS,

called MPS (Figure 10). When analysing a sample, the following measurements has to be performed

when trapping all components. First the tenax of the TDU and PTV has to be blank, to eliminate

components present in the air and on the tenax. When the tenax is blank, the DHS method is started

on a blank sample. A blank sample is an empty glass vial. And the next step is the DHS method for a

sample. These methods will be performed in a sequence.

It starts with placing an empty TDU tube in the TDU and placing an TDU tube with tenax in the tray

on the MPS. The method for making the tenax of the TDU and PTV blank starts (blank TDU).

The following steps will be done by the MPS:

1. Tenax tube will be removed from the TDU and placed in hold

2. Tenax tube (containing contamination) will be removed from the tray and placed in the TDU

Now the desorption step and the injection step starts. After the injection step, the GC/MS starts

to measure.

The next step is performing a DHS analysis of a empty sample (blank DHS). It starts with placing an

empty glass vial in the tray on the MPS.

The following steps will be done by the MPS:

3. Empty sample vial is removed from the tray and placed in the DHS

Incubation step starts.

4. A plastic TDU tube is removed from the DHS carriage and placed in hold in the DHS

5. DHS carriage is placed above the empty glass vial

6. TDU tube with tenax is removed from the TDU and placed in the DHS carriage

Trapping step starts.

7. While the trapping step starts, the empty TDU tube is removed from hold and placed in the

TDU. This is necessary because of the pressure over column. The GC needs to be closed at

any time

8. After the trapping step, the empty TDU tube is removed from the TDU and placed in hold

9. TDU tube with tenax is removed from the DHS carriage and placed in the TDU

The desorption step and injection step starts.

10. While the desorption step starts, the DHS carriage is moved backward and the plastic TDU

tube is placed in the DHS carriage. Also the empty glass vial in placed back in the tray on the

MPS

After the injection step, the GC/MS starts to measure.

The following step is performing a DHS analysis of a sample (DHS analysis of sample). It starts with

filling up the empty glass vial, used in the previous method, with the sample and placing it in the tray

on the MPS.

Steps 3 to 10 will be repeated by the MPS and in this method a glass vial with sample will be used.

After the measurement of the DHS the following steps will be done by the MPS:

11. After the measurement the TDU tube with tenax is removed from the TDU and placed in the

tray

12. The empty TDU tube is removed from hold and placed in the TDU

55

Attachment 2-Figuration of PTV-tube, TDU-tube and sample vial

PTV TDU sample vial

Attachment 3-Chromatograms of contamination of PTV-liner in PTV

1 day

3 days

56

Attachment 4-Chromatograms of contamination of TDU-tube in TDU

1 week

3 days

1 week

57

Attachment 5-Chromatogram of blank DHS

Attachment 6-Chromatograms of contamination of O-ring

2 months

1 min

58

1 day

1 day in aluminium foil

1 week in aluminium foil

59

Attachment 7-Chromatograms of contamination of carrier

Carbohydrate exposed in open environment within Scentarom

Carbohydrate exposed in open environment outside Scentarom

Silica exposed in open environment within Scentarom

60

Attachment 8-Overview of structures of different components

Component Structure Component Structure

diacetyl

citronellal

ethyl acetate

eugenol

cis-3-hexenol

b-ionone

benzaldehyde

g-undecalactone

ethyl hexanoate

thibetolide

phenylethyl alcohol

benzyl salicylate

Silica exposed in open environment outside Scentarom

61

Attachment 9-Parameters of DHS for adjusting split ratio

Range Start

Transfer Heater 50-150 °C 150 °C



10-200 °C 30 °C


0-600 min 1 min








10-200 °C 30 °C




Flow 5-100 ml/min /


20-70 °C /


10-200 °C /

Time / /

Attachment 10-Parameters of DHS for adjusting incubation temperature

Range Start




0-600 min 1 min










Flow 5-100 ml/min /


20-70 °C /


10-200 °C /

Time / /

62

Attachment 11-Chromatograms of analyses for adjusting incubation

temperature

1 2

3 4 5 6

7.I

7.II

8 9

10 11 12

3

4 5

6

7.I

7.II 8 9

10 11 12

3

1 2

7.I

8 9 10 11

6

4 5

7.II

30°C

50°C

65°C

1 2

63

1 2

3 4 5 6

7.I

7.II

8 9

10 11 12

1 2

3

3

4 5

6

7.II

7.I

8

9

10

11 12

75°C

85°C

64

Attachment 12-Parameters of DHS for adjusting incubation time

Range Start




10-200 °C 65 °C







10-200 °C 65 °C




Flow 5-100 ml/min /


20-70 °C /


10-200 °C /

Time / /

Attachment 13-Chromatograms of analyses for adjusting incubation time

0 min

1 2

3 4 5

6

7.I

7.II 8 9

10 11 12

65

1 2

3 4 5 6

7.I

7.II

8 9

10 11 12

1 2

3 4 5

6

7.I

7.II

8 9

10 11 12

15 s

1 min

2 min

1 2

3 4 5

6

7.I

7.II 8 9

10 11 12

66

1 2

3

3

3 4 5

7.II

8

7.II

9

1 2

1 2

4 5

4 5

6

6

6

7.I

7.I

7.I

7.II

10

8 9

11 12

8 9

11 12 10

11 12 10

3

5 min

10 min

15 min

67

Attachment 14-Parameters of DHS for adjusting trapping gas flow

Range Start




10-200 °C 65 °C


0-600 min 5 min







10-200 °C 65 °C



Flow 5-100 ml/min /


20-70 °C /


10-200 °C /

Time / /

Attachment 15-Chromatograms of analyses for adjusting trapping gas

flow

5 ml/min

1 2

3

4 5

6

7.II

7.I

8 9

10 11 12

68

10 ml/min

25 ml/min

50 ml/min

1 2

3

4 5

6

7.I

7.II

8 9

10 11 12

1 2

3

4 5

6

7.I

7.II 8

9

10 11 12

1 2

3 4 5 6

7.I

7.II

8 9

10 11 12

69

Attachment 16-Parameters of DHS for adjusting trapping gas volume

Range Start




10-200 °C 65 °C


0-600 min 5 min




Trapping step Flow (F) 5-100 ml/min 50,0 ml/min



10-200 °C 65 °C



Flow 5-100 ml/min /


20-70 °C /


10-200 °C /

Time / /

100 ml/min

3

4 5

6

7.I

7.II 8

9

10 11 12

1 2

70

Attachment 17-Chromatograms of analyses for adjusting trapping gas

volume

10 ml

50 ml

100 ml

1 2

3

4 5 6

7.I

7.II 8 9

10 11

1 2

3 4 5

6

7.I

7.II

8 9

10 11 12

1 2

3 4 5 6

7.I

7.II

8 9

10 11 12

71

200 ml

150 ml

3

4 5

6

7.I

7.II

8 9

10 11 12

1 2

3 4 5 6

7.I

7.II

8

10

9

11 12

1 2

72

Attachment 18-Parameters of DHS when performing a DHS

Range Start




10-200 °C 65 °C


0-600 min 5 min








10-200 °C 65 °C




Flow 5-100 ml/min /


20-70 °C /


10-200 °C /

Time / /

Attachment 19- Reproducibility of DHS on the relative area of each

component trapped

0

5

10

15

20

25

30

are

a (r

ela

tive

in %

)

Components

Relative area

Test 1

Test 2

Test 3

Test 4

Test 5

Test 6

Test 7

73

Attachment 20-Chromatograms of DHS and SHS comparison

DHS

SHS

1 2

3 4 5

6

7.I

7.II

8 9

10 11 12

1

2

1 2

3

5

4 6

7.I

7.II

8 9

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Dynamic headspace (DHS) technique: set-up and parameter ... · I Acknowledgements I am very grateful for the opportunity that I have received to accomplish this task in Scentarom.

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